Nakamura H et al. (2000), XCS-1, a maternally expressed gene product invo...

XB-ART-10855

J Cell Sci 2000 Jul 01;113 ( Pt 13):2497-505. doi: 10.1242/jcs.113.13.2497.

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XCS-1, a maternally expressed gene product involved in regulating mitosis in Xenopus.

Nakamura H , Wu C , Kuang J , Larabell C , Etkin LD .

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The regulation of the cell cycle during early development is an important and complex biological process. We have cloned a cDNA, XCS-1, that may play an important role in regulating mitosis during early embryogenesis in Xenopus laevis. XCS-1 is a maternally expressed gene product that is the Xenopus homologue of the human cleavage signal protein (CS-1). XCS-1 transcripts were detected in oocytes with the titer decreasing just prior to the MBT. During development the XCS-1 protein was detected on the membrane and in the nucleus of blastomeres. It was also detected on the mitotic spindle in mitotic cells and on the centrosomes in interphase cells. Overexpression of myc-XCS-1 in Xenopus embryos resulted in abnormal mitoses with increased numbers of centrosomes, multipolar spindles, and abnormal distribution of chromosomes. Also, we observed incomplete cytokinesis resulting in multiple nuclei residing in the same cytoplasm with the daughter nuclei in different phases of the cell cycle. The phenotype depended on the presence of the N terminus of XCS-1 (aa 1-73) and a consensus NIMA kinase phosphorylation site (aa159-167). Mutations in this site affected the ability of the overexpressed XCS-1 protein to produce the phenotype. These results suggest that XCS-1 is a maternal factor playing an important role in the regulation of the cell cycle during early embryogenesis and that its function depends on its state of phosphorylation.

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Species referenced: Xenopus laevis
Genes referenced: itprid2 mhc2-dab myc tubg1 xnf7

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	Fig. 1. Alignment of the nucleotide and amino acid sequences of CS-1 proteins and comparison of the conserved sequences of CS-1 among other proteins. (A) The full-length amino acid sequence of XCS-1 was deduced from the nucleotide sequence of the cDNA clone. Identical amino acid sequences of CS-1 proteins (XCS-1; Xenopus cleavage-1; HCS-1; Human CS-1) are highlighted. Highly conserved regions are indicated by arrows. (B) Similarities between XCS-1 and other proteins. Identical amino acid sequences among several proteins are indicated by white letters. Consensus sequences are shown underneath the alignments.
	Fig. 2. Northern and western blot analysis of XCS-1. (A) Approximately 6 mg of total RNA from embryos at various stages were assayed for hybridization to XCS-1 cDNA probe. The samples are from immature oocytes (stage 6), unfertilized eggs, 8 cell (Nieuwkoop-Faber stage 4), 32-cell (stage 6), mid-blastula (stage 8), lateblastula (stage 9), early-gastrula(stage 10), mid-gastrula (stage 11), late-gastrula (stage 12), neural plate (stage 14), mid neural fold (stage 16), neural groove (stage 18) and tailbud (stage 22 and 28) stages. The arrows indicate the XCS-1 transcript (approximately 7 kb) and the position of rRNA. (B) Western blot of embryo extract using affinity purified XCS-1 antibody. The arrow points to the single 55 kDa band.
	Fig. 3. Intracellular distribution of endogenous and exogenous XCS-1 protein. (A-C) Confocal images of whole mounts of stage 10 embryos immunostained using the affinity purified anti-XCS-1 antibody to detect endogenous XCS-1. (A) Arrows are pointing to the XCS-1 protein located on two structures that appear to be centrosomes; (B and C) different focal planes through the same blastomere showing centrosomes on different sides of the nucleus; (D,E,H) in vitro-transcribed RNA from myc-XCS-1 (10 pg) was injected into fertilized eggs. Stage 10 embryos were fixed and sectioned and immunostained with anti-myc antibody or anti-g-tubulin antibody. (D and E) Peroxidase-staining using anti-myc antibody – arrows are pointing to the centrosomal area, the spindle and to the chromosomes on the spindle. (F) Confocal image of endogenous XCS-1 protein decorating intracellular structures as well as cell membrane; (G) indirect immunofluorescence with the anti myc-XCS-1 antibody showing intracellular distribution of exogenous XCS-1 at the centrosomal area of 3 cells (arrows). (H) The same cells in panel G that were immunostained with anti g-tubulin antibodies to show the position of the centrosomes (arrows).
	Fig. 4. Localization of exogenous XCS-1 in mitotic and interphase cells. myc-XCS-1 RNA (10 pg)-injected embryos were fixed and sectioned. Sections were stained with either DAPI (A and B) or antimyc antibodies (red C and D) and detected by indirect immunofluorescence. White arrows point to myc-XCS-1 protein localized on the chromosomes and centrosomes (C) or on centrosomes (D).
	Fig. 5. Overexpressed XCS-1 affects cell division. (A) Stage 8 embryos injected with full-length myc-XCS-1 RNA (50 pg) into a single blastomere of a 2-cell stage embryo. (B) Control stage 6.5 and stage 8 embryos that were injected with myc-XCS-1 (D1-73) mRNA (50 pg) into a single blastomere at the 2-cell stage. (C) Section of a stage 9 embryo injected with myc-XCS-1 RNA (50 pg) into single blastomeres of 2-cell stage embryos showing the effect on cell size. Sections were stained with DAPI to examine the distribution of chromosomes. The large blastomeres toward the bottom of the panel are the injected blastomeres. Arrows are pointing to enlarged blastomeres in A.
	Fig. 6. Overexpression of XCS-1 results in multipolar spindles, extra centrosomes and abnormal nuclei. (A) myc-XCS-1 RNA injected stage 10 embryos that were stained with anti-a-tubulin antibodies to show mitotic spindles and examined by immunofluorescence; (B) DAPI staining of the same section as in A. (A) Extra spindles (arrow); (B) abnormal distribution of chromosomes (arrow); (C and D) DAB color reaction with the anti-a-tubulin antibodies showing multiple spindles and abnormal orientation of the spindle (arrows); (E-G) anti-myc-XCS-1 antibodies showing the location of the myc-XCS-1 protein in a cell from a stage 10 embryo (E); anti-g- tubulin antibody staining (F) and DAPI staining (G) of the same cell showing the multiple centrosomes containing myc-XCS-1 and the presence of two nuclei in different phases of the cell cycle. Arrows point to XCS-1 in E, centrosomes, in F, and nuclei in G; (H) DAPI staining of another cell showing chromosomes in multiple nuclei in different phases of the cell cycle.
	Fig. 7. Fragmented nuclei in XCS- 1 RNA injected embryos. myc- XCS-1 RNA-injected embryos were fixed and analyzed for the distribution of xnf7 protein using anti-xnf7 antibody to show nuclei and spindles. Antibody binding was visualized with FITC-coupled secondary antibody and horseradish peroxidase-coupled secondary antibody and diaminobenzidine staining. (A) myc-XCS-1 RNA injected embryo at stage 10 (FITC). Arrows are pointing to xnf7 staining structures. (B) DAPI staining of the same section as shown in panel A showing that xnf7 staining structures in A contain DNA and represent fragmented nuclei (arrows). (C) mycXCS-1 RNAinjected embryo at stage 10 (peroxidase) showing low magnification view of embryo. Arrows point to affected cells showing abnormal nuclei. (D) is high magnification image of C. (E) Stage 10 embryos injected with XCS-1 (D1-73) RNA (50 pg) into a single blastomere of the 2-cell stage showing normal nuclei stained with anti-xnf7 antibody.
	Fig. 8. Phosphorylation of XCS-1 peptide by immature and mature oocyte extracts. (A) Sequences of peptides used in this study. Numbers correspond to the positions of the amino acid in XCS-1. (B) Wild-type (lanes 1 and 2) and mutant peptides (mutant 1, lanes 3 and 4) (mutant 2, lanes 5 and 6) were used in an in vitro kinase assay with immature oocyte extracts (IOE; lane 1, 3 and 5) or mature oocyte extracts (MOE; lanes 2, 4 and 6).